U.S. patent number 5,364,907 [Application Number 08/071,327] was granted by the patent office on 1994-11-15 for graft copolymers and graft copolymer/protein compositions.
This patent grant is currently assigned to Minnesota Mining and Manufacturing Company. Invention is credited to Patrick L. Coleman, Steven L. Kangas, Thomas A. Kotnour, Richard J. Rolando.
United States Patent |
5,364,907 |
Rolando , et al. |
November 15, 1994 |
**Please see images for:
( Certificate of Correction ) ** |
Graft copolymers and graft copolymer/protein compositions
Abstract
Graft copolymers comprising a poly-alpha-olefin base polymer
selected from the group consisting of polyethylene, polypropylene,
polystyrene, and compatible mixtures thereof, having grafted
thereto an olefinic monomer. The grafted monomer is present in an
amount effective to increase the amount of protein that will bind
to the graft copolymer as compared with the base polymer. Also
disclosed are polymer/protein compositions comprising a graft
copolymer having a protein immobilized on the surface thereof,
processes for the preparation of the above-described graft
copolymers and compositions, methods of immobilizing proteins, and
methods of immunoassay based on such immobilization.
Inventors: |
Rolando; Richard J. (Oakdale,
MN), Coleman; Patrick L. (Minneapolis, MN), Kangas;
Steven L. (Woodbury, MN), Kotnour; Thomas A. (Faribault,
MN) |
Assignee: |
Minnesota Mining and Manufacturing
Company (St. Paul, MN)
|
Family
ID: |
24382562 |
Appl.
No.: |
08/071,327 |
Filed: |
June 2, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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595275 |
Oct 10, 1990 |
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Current U.S.
Class: |
525/54.1;
435/188; 525/193; 525/204; 525/240; 525/241; 525/242; 525/243;
525/244; 525/260; 525/263; 525/265; 525/279; 525/281; 525/296;
525/303; 525/309; 525/327.9; 525/333.3; 525/333.6; 525/375;
525/435; 525/525; 525/530; 525/69; 525/87; 530/403; 530/812;
530/815; 530/816 |
Current CPC
Class: |
C08F
255/02 (20130101); C08F 257/02 (20130101); G01N
33/545 (20130101); B01J 20/28014 (20130101); B01J
20/261 (20130101); B01J 20/264 (20130101); B01J
20/3007 (20130101); B01J 20/321 (20130101); B01J
20/327 (20130101); B01J 20/3276 (20130101); B01J
20/3278 (20130101); Y10S 530/812 (20130101); Y10S
530/815 (20130101); Y10S 530/811 (20130101); Y10S
530/816 (20130101) |
Current International
Class: |
B01J
20/32 (20060101); B01J 20/30 (20060101); C08F
255/00 (20060101); C08F 257/00 (20060101); C08F
257/02 (20060101); C08F 255/02 (20060101); G01N
33/544 (20060101); G01N 33/545 (20060101); C08G
063/48 (); C08G 063/91 (); G08L 009/00 (); G08L
047/00 () |
Field of
Search: |
;525/54.1,69,87,193,204,240,241,242,243,244,260,263,265,327.9,333.3,333.6,375
;435/188 ;530/402,403,811,812,815,816 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0234083 |
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Sep 1987 |
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EP |
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86-302861 |
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Mar 1985 |
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JP |
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0105625 |
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Apr 1988 |
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JP |
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89-367670 |
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Apr 1988 |
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JP |
|
1393693 |
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May 1975 |
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GB |
|
Other References
Plastics Compounding, Jan./Feb. 1986, pp. 44-53 (Eise et al.).
.
Plastics Compounding, Sep./Oct. 1986, pp. 24-39 (Frund et al.).
.
Polymer Prep., 1986, 27, 89 (Sahar). .
Biomedical Applns. of Immobilized Enzymes, vol. 2, T. M. S. Chang,
Ed. Plenum Publishing Corp., (Engvall). .
Clin. Chem. 1976, 22, 1243 (Wisdom). .
J. Colloid and Interface Sci. 1985, 106, 438 (Lavielle et
al.)..
|
Primary Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Griswold; Gary L. Kirn; Walter N.
Busse; Paul W.
Parent Case Text
This application is a continuation of U.S. Patent application Ser.
No. 07/595,275 filed Oct. 10, 1990, now abandoned.
Claims
The claimed invention is:
1. A graft copolymer consisting essentially of a uniform,
homogeneous thermoplastic of
i) polyethylene and
ii) an amount of a grafted monomer selected from the group
consisting of 1-vinylimidazole, hydroxyethyl methacrylate,
polyethylene glycol monomethacrylate, N-vinyl pyrrolidone,
N,N-dimethylacrylamide and mixtures thereof effective to increase
protein binding to the graft copolymer as compared with
polyethylene.
2. The graft copolymer of claim 1 wherein the graft copolymer
comprises about 0.01-20.0 wt. % grafted monomer.
3. The graft copolymer of claim 1 wherein the graft copolymer
comprises about 0.5-10.0 wt. % grafted monomer.
4. The graft copolymer of claim 1 in the form of a microtiter well,
a test tube, a bead, or a film.
5. A graft copolymer consisting essentially of a uniform,
homogeneous thermoplastic of:
i) a base polymer selected from the group consisting of
polypropylene, polystyrene, and mixtures thereof and
ii) an amount of 1-vinylimidazole effective to increase protein
binding to the graft copolymer as compared with protein binding to
only the base polymer, wherein the base polymer is selected from
the group consisting of polypropylene, polystyrene, and mixtures
thereof.
6. The graft copolymer of claim 5 wherein the graft copolymer
comprises about 0.01-20.0 wt. % grafted monomer.
7. The graft copolymer of claim 5 wherein the graft copolymer
comprises about 0.5-10.0 wt. % grafted monomer.
8. The graft copolymer of claim 5 in the form of a microtiter well,
a test tube, a bead, or a film.
Description
BACKGROUND OF THE INVENTION
1. Technical Field
This invention relates to graft copolymers and processes for their
preparation. In another aspect, this invention relates to
immobilization of proteins on synthetic polymers and also to
methods of immunoassay based on such immobilization. This invention
also relates to polymers with proteins immobilized on the surface
thereof.
2. Description of the Related Art
Processing and/or production of polymers using wiped-surface
reactors such as screw extruders and twin-screw extruders is well
known (such processing is often referred to as reactive extrusion).
Twin-screw extruders and their use in continuous processes such as
graft polymerization, alloying, bulk polymerization of vinyl
monomers, and condensation and addition reactions are generally
described in Plastics Compounding, January/February 1986, pp. 44-53
(Else et al.) and Plastics Compounding, September/October 1986, pp.
24-39 (Frund et al.). Graft reactions are said to be carried out by
first melting a polymeric species in the initial stages of an
extruder, injecting a peroxide catalyst into the extruder, and
mixing in a monomer under high shear conditions. Advantages of the
twin-screw extrusion process are said to include narrow
distribution of molecular weight, improved melt-flow properties,
consistent process control, and continuous processing.
Graft polymerization reactions of polyolefins with various monomers
using wiped-surface reactors are known. Such grafting is said to be
useful in providing a polymer adduct with functionality to allow
further modification of structure and properties, and general
mechanistic proposals regarding the formation of these
"mechanochemically synthesized" adducts are discussed in connection
with the grafting of maleic anhydride onto polypropylene in Polymer
Prep.,1986, 27, 89 (Al-Malaika). Particular free radical graft
polymerization reactions have been reported. For example, U.S. Pat.
No. 3,177,270 (Jones et al.) discloses a process of preparing graft
copolymers by malaxing an olefin polymer at a temperature between
110.degree. C. and 250.degree. C. while contacting the polymer with
a minor proportion of a mixture comprising a monovinyl aromatic
compound and optionally one or more other monomers such as acrylic
acid, methacrylic acid, acrylonitrile, methyl methacrylate,
methacrylonitrile, or maleic anhydride, the mixture having
dissolved therein an organic peroxide. British Pat. No. 1,393,693
(Steinkamp et al.) discloses the use of a single-screw extruder to
graft monomers such as maleic anhydride and acrylic acid onto
polyolefins such as polypropylene in the presence of a suitable
free radical initiator such as an organic peroxide. The product
graft copolymers are said to have a melt flow rate (MFR) of at
least 50% greater than the MFR of the base polymer.
U.S. Pat. No. 4,003,874 (Ide et al.) discloses modified polyolefins
obtained by adding an unsaturated carboxylic acid or an anhydride
thereof and an organic peroxide to a polyolefin and melting these
components in an extruder. The polyolefin so obtained adheres to
glass fibers.
U.S. Pat. No. 4,146,529 (Yamamoto et al.) discloses a process for
the production of modified polyolefins by combining a polyolefin
with one or more carboxylic acids or their anhydrides in the
presence of a radical producing agent in an extruder and in the
presence of an organosilane.
U.S. Pat. No. 4,228,255 (Fujimoto et al.) discloses a method for
crosslinking a polyolefin, the polyolefin being a low density
polyethylene or a polyolefin mixture containing a low density
polyethylene, comprising reacting the polyolefin with an organic
silane and an organic free radical initiator to form a
silane-grafted polyolefin, then mixing the silane-grafted
polyolefin with a silanol condensation catalyst. The mixture is
extruded with heating in a single-screw extruder to obtain a
crosslinked polyethylene.
Among the myriad properties of some synthetic polymers is their
ability to reversibly bind proteins. Many techniques for assay of
protein-containing substrates are based on such binding. Enzyme
linked immunosorbent assay, described in "Biomedical Applications
of Immobilized Enzymes", Vol. 2, T. M. S. Chang, Ed. Plenum
Publishing Corp., (Engvall) is but one such technique. ELISA and
other enzyme immunoassay techniques such as those described in
Clin. Chem. 1976, 22, 1243 (Wisdom) generally use a material such
as glass, polycarbonate, or polystyrene as a solid-phase immune
adsorbent, which immobilizes one member of an immunological pair.
The subsequent assay relies on competitive binding of the other
member of the immunological pair in labeled and unlabeled form, to
the immobilized member. One recognized disadvantage of the use of
such techniques is that the immobilized protein is only physically
adsorbed to the immune adsorbent such that adsorbed protein can be
washed off to various degrees by rinsing or contact with aqueous
buffer solutions. A decrease in assay accuracy, precision, and
sensitivity can result from such "leakage" of the adsorbed
protein.
SUMMARY OF THE INVENTION
This invention provides graft copolymers comprising a
poly-alpha-olefin base polymer selected from the group consisting
of polyethylene, polypropylene, polystyrene, and a compatible
mixture of any two or more thereof, having grafted thereto an
olefinic monomer selected from the group consisting of: in the
instance of a polyethylene base polymer, 1-vinylimidazole,
polyethylene-glycol monomethacrylate, and N-vinylpyrrolidone, and a
mixture of any two or more thereof; in the instance of a
polypropylene base polymer, 1-vinylimidazole; in the instance of a
polystyrene base polymer, 1-vinylimidazole; and in the instance of
a base polymer mixture of any two or more of polyethylene,
polypropylene, and polystyrene, 1-vinylimidazole;
the grafted monomer being present in an amount effective to
increase the amount of protein that will bind to the graft
copolymer as compared with the base polymer.
This invention also provides a polymer/protein composition
comprising: a graft copolymer that comprises a poly-alpha-olefin
base polymer selected from the group consisting of polyethylene,
polypropylene, polystyrene, and a compatible mixture of any two or
more thereof, having grafted thereto an olefinic monomer selected
from the group consisting of: in the instance of a polyethylene
base polymer, 1-vinylimidazole, hydroxyethyl methacrylate,
N,N-dimethylacrylamide, polyethyleneglycol monomethacrylate,
N-vinylpyrrolidone, and a mixture of any two or more thereof; in
the instance of a polypropylene base polymer, 1-vinylimidazole; in
the instance of a polystyrene base polymer, 1-vinylimidazole; and
in the instance of a base polymer mixture of any two or more of
polyethylene, polypropylene, and polystyrene, 1-vinylimidazole; the
grafted monomer being present in an amount effective to increase
the amount of protein that will bind to the graft copolymer as
compared with the base polymer, with a protein immobilized on the
surface of said composition.
This invention also provides processes for preparing the graft
copolymers described above. One such process comprises the steps
of:
1) feeding to a reactor materials comprising
(a) the poly-alpha-olefin base polymer
(b) an effective amount of a free radical initiator system
comprising one or more free radical initiators; and
(c) the olefinic monomer; wherein all materials are substantially
free of oxygen;
2) reacting the materials in the reactor to provide a graft
copolymer as described above; and
3) withdrawing the graft copolymer from the reactor.
This invention also provides a method of immobilizing a protein,
comprising the step of:
contacting the protein with a graft copolymer surface, wherein the
graft copolymer comprises a poly-alpha-olefin base polymer selected
from the group consisting of polyethylene, polypropylene,
polystyrene, and a compatible mixture of any two or more thereof,
having grafted thereto an olefinic monomer selected from the group
consisting of: in the instance of a polyethylene base polymer,
1-vinylimidazole, hydroxyethyl methacrylate,
N,N-dimethylacrylamide, polyethyleneglycol monomethacrylate,
N-vinylpyrrolidone, and a mixture of any two or more thereof; in
the instance of a polypropylene base polymer, 1-vinylimidazole; in
the instance of a polystyrene base polymer, 1-vinylimidazole; and
in the instance of a base polymer mixture of any two or more of
polyethylene, polypropylene, and polystyrene, 1-vinylimidazole, the
grafted monomer being present in an amount effective to increase
the amount of protein that will bind to the graft copolymer as
compared with the base polymer, at a temperature and for a time
sufficient to cause the protein to become immobilized on the
surface.
Further, the invention provides a method of immunoassay comprising
the steps of:
1) treating an article comprising a surface of a polymer/protein
composition as described above with one member of an immunological
pair;
2) incubating the treated article with a solution suspected of
containing the second member of the immunological pair; and
3) determining the amount of the second member of the immunological
pair present in the solution.
By virtue of the grafted monomers, graft copolymers of the
invention provide an increased amount of irreversible binding
(i.e., immobilizing) of proteins for the purposes of, e.g.,
immunoassay. Accordingly, graft copolymer/protein compositions of
the invention allow improvement in bioassay accuracy, precision,
and sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an exemplary flow diagram of a process for preparing the
graft copolymers of the invention and those used in the
compositions of the invention. Ancillary equipment known to those
skilled in the art, such as pumps and valves, has not been
illustrated, and secondary process streams such as utility lines
(e.g., cooling water) have been omitted.
FIG. 2 is a flow diagram of a counter-rotating twin screw extruder
useful in preparing graft copolymers.
DETAILED DESCRIPTION OF THE INVENTION
A graft copolymer of the invention comprises a poly-alpha-olefin
base polymer and a monomer grafted thereto via an alkenyl group.
The double bond is of course not present in the product graft
copolymer; rather, in the grafting process the alkenyl group
becomes a saturated (e.g., alkylene) link between the base polymer
and the grafted moiety. In the instant specification and claims a
reference to a grafted alkenyl group designates such a saturated
link and does not designate the presence of olefinic unsaturation
in the grafted monomer as it is incorporated in the graft
copolymer.
Suitable base polymers include poly-alpha-olefins selected from the
group consisting of polyethylene, polypropylene, polystyrene, and a
compatible mixture of any two or more thereof. Base polymers of
virtually any molecular weight are suitable. Likewise, base
polymers and compatible mixtures thereof with a wide range of melt
index values (e.g., from about 0.1 to about 1500) are suitable.
The olefinic monomer is selected from the group consisting of: in
the instance of a polyethylene (PE) base polymer, 1-vinylimidazole
(VIm), a polyethylene glycol monomethacrylate (PEG;
polyethyleneglycol monomethacrylates of virtually any molecular
weight, e.g., in the range from about 200 to about 10,000 are
suitable), N-vinylpyrrolidone (NVP), and a mixture of any two or
more thereof; in the instance of a polypropylene (PP) base polymer,
1-vinylimidazole; in the instance of a polystyrene (PS) base
polymer, 1-vinylimidazole; and in the instance of a base polymer
mixture of any two or more of polyethylene, polypropylene, and
polystyrene, 1-vinylimidazole.
A graft copolymer of the invention comprises an amount of the
grafted monomer effective to increase the amount of protein that
will bind to the graft copolymer as compared with the base polymer.
Stated another way, the graft copolymer binds proteins to a greater
degree than does the base polymer. The amount that constitutes an
effective amount of the grafted moiety will depend upon the
particular grafted monomers and the particular base polymer.
Generally, however, a graft copolymer comprises about 0.01% to
about 20%, preferably 0.5 to about 10% by weight of grafted
monomer. In the preparation of the graft copolymers (described in
detail below) it is preferred to use like quantities of monomer,
i.e., preferably about 0.01 to about 20% or more by weight, more
preferably 0.5 to about 10% by weight based on the weight of the
base polymer.
In a graft copolymer/protein composition of the invention,
different grafted monomers are suitable depending on the particular
base polymer. In the instance of a polyethylene base polymer, the
grafted monomer is selected from the group consisting of
1-vinylimidazole, hydroxyethyl methacrylate (HEMA),
N,N-dimethylacrylamide (DMA), polyethyleneglycol monomethacrylate,
N-vinylpyrrolidone, and a mixture of any two or more thereof. In
the instance of a polypropylene or polystyrene base polymer, and in
the instance of a compatible base polymer mixture of any two or
more of polyethylene, polypropylene, polystyrene, the grafted
monomer is 1-vinylimidazole.
A graft copolymer used in a graft copolymer/protein composition of
the invention, like a graft copolymer of the invention, comprises
an amount of the grafted monomer effective to increase the amount
of protein that will bind to the graft copolymer as compared with
the base polymer. The amount that constitutes an effective amount
is as discussed above in connection with graft copolymers of the
invention.
In order to prepare a graft copolymer, the base polymer and the
monomer are reacted in the presence of an initiator system
comprising one or more free radical initiators. The initiator
system serves to initiate free radical grafting of the monomer. In
a process involving a base polymer that does not undergo
substantial crosslinking under polymer melt conditions in the
presence of a free radical initiator, the base polymer is degraded
in the reactor. However, the selection of an appropriate initiator
system affords a product graft copolymer that better retains the
molecular weight of the base polymer.
Many initiators are known. Suitable initiators include:
hydroperoxides such as cumene, t-butyl, and t-amyl hydroperoxides,
and 2,5-dihydroperoxy-2,5-dimethylhexane; dialkyl peroxides such as
di-t-butyl, dicumyl, and t-butyl cumyl peroxides,
2,5-dimethyl-2,5-di(t-butylperoxy)hexane, and
2,5-dimethyl-2,5-di(t-butylperoxy)hex-3-yne; peroxyesters such as
t-butyl perbenzoate and di-t-butyl-diperoxy phthalate, diacyl
peroxides such as benzoyl peroxide and lauroyl peroxide;
peroxyketals such as n-butyl-4,4-bis(t-butylperoxy)-valerate and
1,1-di-t-butylperoxy-3,3,5-trimethylcyclohexane; and azo compounds
such as azoisobutyronitrile.
The reaction conditions under which a graft copolymer is prepared
typically involve heating at about 150.degree. C. to about
250.degree. C. The reactants typically have a residence time of
about 1 to about 20 min. It is therefore difficult to select a
single initiator with a decomposition rate such that initiating
radicals will be present in a substantial concentration for a
prolonged period of time when a relatively low concentration of
initiator is used. It is therefore preferred to use a mixture of at
least two initiators as an initiator system.
Proper selection of the components of the initiator system
overcomes the above-discussed difficulty with single initiators,
and allows control and optimization of the physical properties of
the product graft copolymer. Generally it is preferred that each
initiator in an initiator system have a rate of decomposition
substantially different from those of the other initiators in the
initiator system. For example, in a process with a residence time
of about 5-10 minutes at a temperature of about 200.degree. C., an
initiator system wherein one initiator has a half-life of about 30
seconds and the other initiator has a half-life of about 2 minutes
has been found to be suitable.
Preferred initiator systems include mixtures comprising from about
40% to about 60% by weight of
2,5-dimethyl-2,5-di(t-butylperoxy)hexane, (such as that
commercially available as LUPERSOL.TM. 101 from Pennwalt
Corporation) and from about 60% to about 40% by weight of an
initiator such as 2,5-dimethyl-2,5-di(t-butylperoxy)hex-3-yne,
(such as that commercially available as LUPERSOL.TM. 130 from
Pennwalt Corporation), t-butylhydroperoxide, or di-t-butylperoxide.
Initiator decomposition rates are temperature dependent, and other
particular initiator systems and preferred concentration thereof
can be selected by those skilled in the art consistent with the
temperature of the reaction and the residence time of the
reactants.
The total initiator concentration is preferably from about 0.1% to
about 1%, more preferably from about 0.25% to about 0.5% based on
the weight of the base polymer.
The graft copolymers can be prepared using various well known
reactors such as stirred tank reactors, tubular reactors, and
extruders. The graft copolymers are preferably made by a process
involving a wiped-surface reactor. A wiped-surface reactor
comprises a shell or vessel that contains at least one rotor having
a wiping portion located close to the inside surface of the shell
and a root portion that is spaced substantially further from the
shell than the wiping portion. As the rotor is rotated, the wiping
portion passes close enough to the inside surface of the shell to
clean the surface and form a seal when the reactor contains monomer
and/or polymer but not so close as to cause permanent deformation
of either the rotor or shell. It is necessary that the root surface
of the rotor also be wiped or cleaned continuously during the
operation of the reactor.
Intermeshing twin screw extruders can be used as wiped-surface
reactors. The screws function as the rotors and the flight lands
function as the wiping portion, while the screw root surface
between the flight lands functions as the root surface. Clearances
between the inside of the barrel wall of the extruder and the
flight lands of the screws are preferably in the range of about
0.25 to 0.5 mm. Although co-rotating twin screw extruders can be
used, counter-rotating twin screw extruders are preferred. The
counter-rotating extruder acts as a positive displacement pump
conveying the reactant stream, and it also behaves like a series of
small mixing zones or continuous stirred tank reactors. The
counter-rotating twin screw extruder also gives good control over
melting, mixing, and reaction temperatures.
Preferably, the screws of a counter-rotating twin screw extruder
are divided into segments, i.e., the extruder screws can be
composed of a number of separate screw segments that fit onto a
common drive shaft by means of a keyway and can be disassembled and
rearranged in various orders and configurations. It is also
possible to utilize screw segments having multiple (e.g., two or
three) starts and various pitch, and one or more screw segments can
be reversed in order to increase mixing. Residence time of the
reactants, and thus the properties of the resultant product, can
therefore be varied by selection of screw pitch and/or screw speed
(i.e., screw rpm). Furthermore, each particular zone of a twin
screw extruder can be independently heated or cooled by external
heating or cooling means, allowing further control of reaction
conditions.
The use of a wiped-surface reactor is discussed with reference to
FIG. 1. The base polymer can be fed in a region of the reactor
coincident with the region in which the initiator system is fed.
For example, the desired base polymer, preferably in pellet form,
can be wetted with a free radical initiator system and purged with
an inert gas such as nitrogen, helium, argon or the like, to render
the material substantially free of oxygen (i.e., oxygen, if
present, is present in an amount such that it does not
significantly affect the desired free radical polymerization
reactions). It is preferred to carry out the reaction under
anhydrous conditions.
The base polymer/initiator mixture can be fed at a predetermined
rate into feed zone 1 of the wiped-surface reactor. It is
preferred, however, to feed the base polymer in a region of the
reactor prior to or coincident with the region in which the
initiator system is fed. Preferably, in instances where the base
polymer is a poly-alpha-olefin that does not undergo substantial
crosslinking under polymer melt conditions in the presence of a
free radical initiator, the base polymer is fed to the reactor in a
region of the reactor preceding or coincident with the region in
which the initiator system is fed, and the monomer is fed to the
reactor in a region of the reactor subsequent to the region in
which the initiator is fed. In instances where the
poly-alpha-olefin base polymer undergoes substantial crosslinking
under polymer melt conditions in the presence of a free radical
initiator, the base polymer and the initiator are preferably fed to
the reactor in a region preceding the region in which the monomer
is fed, but at a temperature such that crosslinking of the base
polymer is minimized or prevented prior to the addition of the
monomer.
The feed zone 1 typically comprises a feed throat, into which the
base polymer can, if desired, be fed into the upstream end, and
into which the initiator system can be fed at the downstream end. A
further alternate method of feeding the base polymer and the
initiator involves the use of a 2-component feed zone consisting of
a base polymer feed zone into which the base polymer is fed,
followed in sequence by a separate initiator feed zone into which
the initiator is fed. The extruder is preferably starve fed, i.e.,
all material fed into the feed zone is conveyed into
initiation/melt zone 2 of the extruder, and nothing is held up in
the feed zone 1. Feed rates can vary with the size of the reactor
and for any given size of reactor, one skilled in the art will be
able to determine suitable feed rates. As an example, when a
LEISTRITZ.TM. 34 mm counter-rotating twin screw extruder is used
feed rates are preferably from about 0.4 Kg/h to about 9 Kg/h. The
feed zone screw preferably has a high pitch (e.g., 20 mm) to
accommodate base polymer pellets. The feed zone can, if desired, be
operated in a temperature controlled manner, depending on the
reactants, reaction conditions and the like. Generally, it is
suitable to maintain the feed zone of the extruder in a temperature
range from about 10.degree. C. to about 50.degree. C., depending on
the base polymer used.
In initiation/melt zone 2, the initiator system and the base
polymer are mixed and heated. When non-crosslinking base polymers
such as polypropylene and polystyrene are used, the temperature is
preferably such that radical chain reactions are initiated.
Preferred temperatures will depend on the particular base polymer
and initiator system, but generally temperatures in the range
between 150.degree. C. and about 250.degree. C. are suitable. When
crosslinking base polymers such as polyethylene are used, both the
feed zone and the initiation/melt zone are preferably kept at a
temperature such that the initiator does not produce initiating
radicals at a significant rate. As the residence time of the
materials in these zones is only a small fraction of the total
residence time, this serves to minimize or prevent the crosslinking
of the base polymer prior to addition of the monomer. Again
preferred temperatures will depend on the particular base polymer
and initiator system. Generally, however, temperatures between
about 100.degree. C. and 150.degree. C. are preferred.
In monomer addition zone 3, a nitrogen-purged monomer is added,
usually by means of a high pressure pump and under an inert
atmosphere. The monomer is generally fed as a liquid or as a
solution in an inert solvent (e.g., decane, toluene,
tetrahydrofuran or the like). Again, feed rates are variable, and
when a LEISTRITZ.TM. 34 mm counter-rotating twin screw extruder is
used, feed rate is preferably about 4 g/h to about 180 g/h. It is
preferred to maintain the monomer addition zone at a temperature of
about 150.degree. C. to about 250.degree. C.
Grafting proceeds in reaction zone 4. The reaction zone is heated.
The preferred temperature will depend on the particular base
polymer and initiator system used. Further, the preferred
temperature of the reaction zone will depend on the intended
residence time in the reaction zone. Generally, temperatures in the
range of 150.degree. C. to 250.degree. C. and residence times in
the range of 1 minute to 10 minutes are suitable.
In reactions where there remains residual monomer, it is preferred
to remove the residual monomer from the product by venting. This
can be done in devolatilization zone 5, where a vacuum (e.g., about
10 kPa absolute pressure) can be applied to a vent line. The
resultant product is passed through block zone 6, which conveys the
product graft copolymer for any further processing as desired,
e.g., shaping in a die, extruding, quenching in a suitable
quenching liquid, or pelletizing to useful dimensions for
convenience of handling and/or storage.
In instances where it is desirable to quench the graft copolymer in
a quenching liquid, any suitable quenching liquid can be used.
Water is commonly used. However, quenching in water can cause some
undesirable hydrolysis of grafted hydrolytic moieties (if any),
such as esters that will be present in graft copolymers wherein
polyethyleneglycol monomethacrylate or hydroxyethyl methacrylate
are the grafted monomers. Further, quenching in water can cause the
graft copolymer to have a relatively high moisture content, which
can cause internal hydrolysis of hydrolytic groups (if any) and
poor performance of the graft copolymer upon molding. Therefore, it
is preferred to quench the graft copolymer in a quenching liquid
that is inert to any functional groups present in the monomer. It
is also desirable for such a quenching liquid to have low
volatility and a high specific heat. Suitable quenching liquids can
be easily selected by those skilled in the art. Particularly
preferred quenching liquids include inert liquid fluorocarbons.
A graft copolymer surface can bind (i.e., immobilize) proteins. The
protein can be, for example, an antibody such as anti-human IgE, a
protein such as Protein A, or an enzyme. Preferred proteins for
immobilization include those with a molecular weight of at least
1000, most preferably at least about 4000.
A graft copolymer can be prepared, for example, in the form of an
article such as a microtiter well or a test tube or in the form of
beads or a film. To bind (i.e., immobilize) a protein to the
surface of the article, the article can be contacted, e.g.,
incubated, with a protein, e.g., a serum or other solution
containing a protein. The protein can also, if desired, contain a
trace level of labeled (e.g., radiolabeled or fluorescence-labeled)
protein to allow assay of the protein. An article with a protein
bound thereto can then be further incubated, for example, with a
relatively concentrated second protein solution such as bovine
serum albumin, to block any remaining surface of the article and to
displace initially adsorbed protein from the surface of the
article.
An article treated as described above can be treated (e.g.,
incubated) with a protein denaturing agent such as sodium
dodecylsulfate (SDS) to remove loosely-bound protein from the
surface. Analysis of the resulting article shows that the amount of
protein that is retained on the graft copolymer surface is
increased by the grafted moiety.
The increased amount of irreversible binding of proteins such as
antibodies in the graft copolymer/protein compositions of the
invention suggests utility in applications where protein
immobilization is desirable, e.g., diagnostic applications in which
proteins are immobilized, including microtiter well assay devices,
bead suspensions, and the like for use in ELISA and other well
known enzyme immunoassay techniques such as those described in
Clin. Chem. 1976, 22, 1263 (Wisdom). Furthermore, it is known that
a proteinaceous layer will promote binding of cells to hydrophobic
and hydrophilic base polymers. This invention allows one to
immobilize proteins such as albumins, collagens, basement membrane
fractions, etc., or specific proteins such as fibronectin, laminin,
monoclonal antibodies, or adhesion proteins, etc., all of which can
promote binding of cells to a polymer surface.
The immobilization of a protein on a graft copolymer can be carried
out by contacting the protein with a graft copolymer surface at a
temperature and for a time sufficient to cause the protein to bind
to the graft copolymer surface. While it is not practical to
enumerate particular conditions suitable for each and every
protein, such conditions can be easily selected by those skilled in
the art. Generally, however, room temperature exposure of a graft
copolymer surface to a solution of the protein in an appropriate
solvent will be suitable to bind the protein to the surface.
The amount of grafted moieties on the surface of a graft copolymer
can be measured by conventional means such as x-ray photoelectron
spectroscopy, Fourier transform infrared spectrophotometry,
attenuated total reflectance infrared spectrophotometry, and the
like.
The following describes the preparation of graft copolymers and
graft copolymer/protein compositions. Temperatures are in degrees
Celsius, and all parts and percentages are by weight. Graft
copolymers are designated herein by enumerating the base polymer
and the grafted monomer, e.g., the designation PE/DMA represents a
graft copolymer comprising a polyethylene (PE) base polymer having
N,N-dimethylacrylamide (DMA) grafted thereto.
Intermediate A
Preparation of polyethylene (PE)/hydroxyethyl methacrylate
(HEMA).
HEMA was grafted onto linear low-density PE (DOWLEX.TM. 2517, melt
index:25, Dow Chemical Co.,.Midland, MI) using a counter-rotating
34 mm LEISTRITZ.TM. twin-screw extruder model LSM 30.3466,
(Nuremburg, Germany), with a length:diameter ratio at 35:1,
configured as described below with reference to FIG. 2.
FIG. 2 shows a twin-screw extruder with a feed hopper 10, feed zone
12, and a heated barrel that comprises: an initiation/melt zone
comprising barrel section 14; a reaction zone comprising a monomer
feed zone (barrel section) and barrel sections 18, 20, 22, 24, and
26; a devolatilization zone comprising barrel section 28; and a
block zone comprising barrel sections 30 and 32. Each barrel
section is 120 mm long, and the extruder has a total length of 1200
mm.
Transducer ports (e.g., T4 represents transducer number 4 located
in barrel section 24) are located at 30 mm, and/or 90 mm into each
heated barrel section. Thermocouple ports are located at 60 mm into
each heated barrel section.
The polyethylene base polymer was directly fed into the feed
throat. A 1:1 mixture by weight of LUPERSOL.TM. 101 and
LUPERSOL.TM. 130 was fed at 4.1 mL/h, and a 1:1 mixture by weight
of LUPEROX.TM. 500 dicumyl peroxide (Pennwalt) and decane was also
fed at 4.2 mL/h, each to the feed throat and each by a separate
nitrogen purged RUSKA.TM. pump. The HEMA was purged with nitrogen,
added to a nitrogen-purged RUSKA.TM. positive displacement pump,
and added at a rate of 160 mL/h in heated barrel section 16, 270 mm
from the start of the screws. Total flow rate was 40 g/min. Screw
speed was 103 rpm. The temperature profile was as follows: Section
14, 253.degree.; Section 16, 145.degree.; Section 18, 181.degree.;
Section 20, 199.degree.; Section 22, 201.degree.; Section 24,
197.degree.; Section 26, 205.degree.; Section 30, 234.degree.;
Section 32, 205.degree.; Section 34, 202.degree. . In heated barrel
section 28 residual monomer was removed by vacuum. The product
graft copolymer was conveyed from the block zone (barrel sections
30 and 32), into a water bath and fed into a Conair Co. (Bay City,
Michigan) pelletizer to afford generally cylindrical beads of 3-4
mm in length and about 1 mm in diameter. The melt index of the
product graft copolymer was 18 as measured by ASTM D-1238,
indicating that crosslinking of the polyethylene occurred during
the grafting process.
Intermediate B and Examples 1-3
Preparation of PE/NVP (Intermediate B), PE/VIm, PP/VIm, and
PE/PEG.
PE (DOWLEX.TM. 2517) and PP (DYPRO.TM. 8771) were independently
used as base polymers, and NVP, polyethyleneglycol monomethacrylate
(SIPOMER.TM. HEM-10 Alcolac), and VIm (Aldrich Chem. Co.) were
independently used as monomers. Graft copolymers were prepared in a
LEISTRITZ.TM. 34 mm twin-screw extruder as generally described
above in connection with Intermediate A. The monomer was purged
with nitrogen gas for 15-30 min prior to use. The feed hopper and
feed throat of the extruder were kept under nitrogen gas
throughout. The monomer was injected into the second zone of the
extruder via a RUSKA.TM. single-piston positive-displacement pump
at a pressure of 50 psi. The initiator (a 1:1 mixture of
LUPERSOL.TM. 101 and LUPERSOL.TM. 130) was fed into the open feed
throat via a dual-piston RUSKA.TM. pump. The extruded graft polymer
was formed into a strand, quenched, and pelletized. Other
conditions are listed in Table 1 below.
TABLE 1 ______________________________________ Polymerization
Conditions Graft Copolymer PE/VIm PP/VIm PE/NVP PE/PEG Condition
Ex. 1 Ex. 2 Int. B Ex. 3 ______________________________________
Screw speed (rpm) 85 100 100 100 Temperature (.degree.C.) Section
14 154 195 153 150 16 150 199 147 150 18 165 204 157 160 20 163 200
159 160 22 160 197 157 160 24 154 195 153 160 26 158 202 161 160 28
185 215 184 160 30 161 204 163 160 32 161 182 163 160 Polymer flow
(g/min) 39 42.1 42.9 42 Init. flow (mL/h) 3.0 8.9 3.0 8.9 Initiator
Percent 0.1 0.3 0.1 0.3 Monomer flow 30 120 120 160 (mL/h) Percent
Incorporated 0.17 3.2 1.9 3.1 Monomer
______________________________________
Intermediate C
Preparation of PE/DMA.
A graft copolymer was prepared in a Brabender Plasti-corder
reactor, type EPL-V5501, (C. W. Brabender Co.), equipped with a
type RE.E.6 mixing head, a type SP-T1002 temperature control
console, and a torque rheometer. The reactor was preheated to
180.degree. C. under a nitrogen purge. PE (DOWLEX.TM. 2517) was
used as base polymer. N,N-Dimethylacrylamide (DMA, Aldrich Chemical
Co.) was used as the monomer. The base polymer (45 g) was added to
the reactor and mixed at 30 rpm until fully melted. The initiator
(0.14 g of a 1:1 mixture of LUPERSOL.TM. 101/LUPERSOL.TM. 130) was
added to the polymer and allowed to mix for about one-half minute.
DMA (2.4 g; 5 weight percent) was added to the mixture and allowed
to react for 3 minutes. The mixture was-removed from the reactor
and cooled to ambient temperature. The PE/DMA contained 0.25% DMA
by weight. Cooled samples were stored in plastic bags until
use.
Example 4
Preparation of PS/VIm.
According to the general method of Intermediate C, polystyrene (39
g, Polysar TM 101-300, melt index 2.2, Polysar Inc., Leominster,
Massachusetts) and 1-vinylimidazole (1.0 g) were reacted to afford
PS/VIm.
Examples 5-11
The immobilization of protein on PE, PP, PS, and their graft
copolymers.
Film samples of the graft copolymer with thickness of about 0.13 mm
were made by pressing (at a pressure of about 41.4 kPa for 30
seconds using a WABASH.TM. heated press, Wabash, Indiana) about 10
g of the graft copolymer between TEFLON.TM. plates at about
200.degree. C. Pressed samples were quenched from the molten state
to the solid state in a room temperature water bath and cut into
discs of 8 mm diameter using a conventional paper punch.
Recombinant Protein A (rProtA), purchased from Repligen (Cambridge,
Massachusetts), was radioiodinated using Iodo-beads.TM. (Pierce
Chemical Co., Rockford, Illinois). rProtA (200 .mu.L of 250
.mu.g/mL), with a specific radioactivity of 2000 cpm/.mu.g of
protein, was incubated with triplicate samples of 8 mm film discs
in 25 mM sodium phosphate, pH 7.5, with 150 mM sodium chloride.
Incubations were terminated after 2 h by removal of the solution
followed by addition of 500 .mu.L of 1.0 M ethanolamine, pH 9.0,
for 1 h. Finally, the film discs were rinsed three times with the
chloride-phosphate buffer for 45 minutes, then transferred to a
clean tube for radioactivity determination using a Packard Model
5230 Gamma Scintillation Spectrometer (Packard Instrument Co.,
Downers Grove, Illinois). The film discs were subsequently
incubated for 4 h at 37.degree. with an aqueous solution of 1% w/v
sodium dodecylsulfate (SDS), rinsed three times with the same SDS
solution, followed by a final radioactivity determination.
TABLE 2 ______________________________________ Comparative Binding
of Protein on Control and Grafted Polymers Adsorbed Tightly Bound
Protein SDS Protein Example Polymer (.mu.g/cm.sub.2) Resistance
(.mu.g/cm.sub.2) ______________________________________ PE 0.51 22%
0.11 5 PE/VIm 1.12 40 0.46 6 PE/HEMA 0.77 32 0.25 7 PE/DMA 0.88 27
0.23 8 PE/NVP 0.81 31 0.24 PE* 0.44 19 0.08 9 PE/PEG* 0.54 53 0.29
PP 0.90 24 0.22 10 PP/VIm 1.04 28 0.29 PS* 1.40 28 0.39 11 PS/VIm*
2.34 50 1.16 ______________________________________ *Protein A
solution had specific radioactivity of 2250 cpm/.mu.g
The amount of protein bound to the base polymers increased in the
order of the increasing hydrophobicity of the base polymer, i.e.,
PS >PP >PE. All grafted monomers enhanced the binding of
protein relative to base polymer surfaces. All the graft copolymers
had increased SDS resistance compared with the base polymer.
Examples 12-16
Stability of protein adsorbance in the presence of blood
proteins.
Protein incubation was carried out as described in Examples 5-9
above. The specific radioactivity of rProtA was 1360 cpm/.mu.g.
After the initial radioactivity determination the films were
incubated for 7 days at ambient temperature with 500 .mu.L of a 1:1
buffer:human serum solution. Residual radioactivity was determined
following the incubation, aspiration of the serum solution, and two
buffer rinses. The films were subjected to the SDS treatment
described in Examples 5-9, and a final radioactivity determination
was made. The results are set forth in TABLE 3 below.
TABLE 3
__________________________________________________________________________
The Effect of Long-Term Incubation of Protein-Bound Films in Serum
Adsorbed Post-plasma SDS Covalent Example Polymer Protein
Adsorbance Resistance Protein
__________________________________________________________________________
PE 0.64 .mu.g/cm.sup.2 0.22 .mu.g/cm.sup.2 6% 0.04 .mu.g/cm.sup.2
12 PE/VIm 1.60 0.89 33 0.53 13 PE/HEMA 1.06 0.48 19 0.20 14 PE/DMA
1.04 0.41 6 0.07 15 PE/NVP 1.08 0.34 6 0.06 PP 1.18 0.39 10 0.12 16
PP/VIm 1.45 0.66 15 0.23
__________________________________________________________________________
Treatment with plasma proteins followed by SDS treatment is a
stringent test to remove proteins from a surface. Surprisingly,
three of the polymers (PE/VIm, PE/HEMA, PP/VIm) contained residual,
tightly-bound protein.
* * * * *